Ion Channel Mechanisms Underlying Spatiotemporal Plasticity Of Primary Sensory Neuronal Action Potentials

Information from exogenous and endogenous environments can be transducted, encoded, integrated,stored and transmitted in the nervous system. The processes are completed through the generation and propagation of"all or none"action potentials with regular or irregular activities on cell bodies, axonal nerve fibers as well as trans-synaptic transmission. In general, the phenomenon of"all or none"means that amplitude of action potential (AP) should be constant under different stimulus intensities and distances. The property of"all or none"ensures the accuracy of information. At present, there are many theories on the information encoded by trains of APs, such as mean rate code, population code, temporal code, pattern code etc. However, the correlation between firing pattern and neuronal information remains controversial for many years. A recent study suggested that the patterns of neuronal activities in cortex might not through accurate firing patterns. The results seriously challenged the popular theory on neuronal rhythmic activity-encoded information. Is there any other means to transmit neuronal information beyond rhythmic patterns? It's a poorly known field.At present, noxious information seems to be mediated by different AP trains generated from nociceptors expressed in cell body and nerve terminal of primary sensory neurons, that can preliminarily encode the information of stimulus (such as pattern, intensity and duration). The latter could be transmitted to spinal cord along the central terminals of somatic sensory fiber in certain time course. Different from the classical AP, the generation and function of APs in nociceptor are poorly understood because of the variabilities and modalities of ion channels,lack of specific ion channel blockers and complex modulations of channel functions. Importantly, because of the theory of information encoded by AP with"all or none"property for a long time, there's very little investigation on the characterization and significance of single AP waveform (especially amplitude and depolarization duration) during different pathologic-physiological conditionsIn present study, we investigated the characterization of AP waveform generated in dorsal root ganglion (DRG) neurons, the first ones in the series of neurons that lead to pain sensation. By using the patch clamp recording, we recorded and analyzed the spatial parameter (amplitude) and temporal parameters (depolarization duration and repolarization duration) of DRG neuronal AP waveform under different in vitro and in vivo levels. The former were performed in the situation of disequilibrium of calcium homeostasis whereas the latter were performed in the situation of inflammatory pain. Furthermore, we investigated the functions and significance of two tetrodotoxin-resistant (TTX-R) sodium channels, NaV1.8 and NaV1.9, in the neuronal AP plasticity. Through researching on the single AP of DRG neurons, we got the following results.1. Action potentials generated in primary sensory neuron perform spatiotemporal plasticityUnder normal situation and without any stimulation, APs produced in spontaneous discharging DRG neuron are"all or none"and having identical spatial parameter parameters and temporal parameters. APs in DRG neuron generated by 10 identical electrical stimulation (intensity, 300 pA; duration, 3 ms; interval, 200 ms) are"all or none". APs in DRG neuron generated by 10 different electrical stimuli (intensity, 50-500 pA; duration, 3 ms; interval, 200 ms) are quite different in spatial and temporal parameters. To understand the characterizations of neuronal AP evoked by electrical stimulation, we apply a series of different electrical stimuli(intensity, 50-500 pA;△=50 pA, duration,0.5-5 ms; interval, 200 ms; number of trains, 10; total number of stimuli, 100 ) . We found the following characterizations: 1) Under certain temporal parameters,AP amplitude is not changed by duration increment but enhanced by intensity increment. 2) Under superthreshold stimulus intensitity, total duration of AP is initially shortented, then gradually recovered, whereas under the same stimulus duration, the values are shortened by stimulus intensity increment. 3) Under the same superthreshold stimulus intensitity, depolarization duration of AP is not changed, whereas the value is shortened by stimulus intensity increment. 4) Under the same superthreshold stimulus intensitity, repolarization duration of AP is initially shortented, then gradually recovered, whereas under the same stimulus duration, the values are shortened by stimulus intensity increment. These results indicated that AP generated in DRG neuron was plasticity. In addition, the plasticity of AP could be changed parameters of electrical stimuli. Intensity of electrical stimulation mainly affected AP amplitude and depolarization duration, while the duration of electrical stimulation mainly affected AP repolarization duration.2. Calcium homeostasis is required for the maintainance of"all or none"property in primary sensory neuron1) Effects of extracellular calcium change on AP of DRG neuron. After whole-cell performed, we recorded electrical stimuli-evoked APs (intensity, 500 pA; duration, 3 ms) under 2.5 Ca,0 Ca,1 Ca,5 Ca and 10 Ca in bath solution, respectively. Under 0 Ca condition, the AP amplitude was significantly decreased, total duration and repolarization duration were lengthened,whereas the depolarization duration was not changed. The suppressive effects might through membrane potential fluctuation because 0 Ca caused 10 mV resting membrane potential (RMP) change shifting to depolarization forward. In contrast, 5 Ca and 10 Ca had nearly no effects on AP spatial and temporal parameters,in addition to lengthen the depolarization duration.2) Effects of intracellular calcium decrease on AP of DRG neuron. After pre-incubating neurons with thapsigargin (1μM )for 15 min to delete intracellular calcium,we recorded electrical stimuli-evoked APs (intensity, 500 pA; duration, 3 ms). Comparison with the 2.5 Ca condition, intracellular calcium decrease significantly inhibited AP amplitude generated with 500 pA stimulation, lengthened total duration repolarization duration, but had no effects on depolarization duration.3) Effects of intracellular calcium increase on AP of DRG neuron. At first, we used combined voltage clamp and calcium imaging test to clarify parameters of capsaicin-induced responses, including duration and amplitude of intracellular calcium increase, duration of DRG neuronal membrane potential and inward current shifting. The peak reaction of intracellular calcium mobilization induced by capsaicin(0.5μM, 10 s duration)perfusion appeared at 10 s after drug adminidtration, while the duration of calcium response lasted more than 120 s. Duration of inward current and membrane potential shifting could return to baseline level within 30 s. IP3 receptor blocker, 2-APB(50μM)can not block capsaicin-induced responses. Ryanodine receptor blocker,ruthenium red (10μM)completely block capsaicin-induced calcium response and significantly suppressed capsaicin-induced inward current. Then, we recorded AP in DRG neurons induced by electrical stimuli (intensity, 500 pA; duration, 3 ms) under normal solution and after capsaicin perfusion. The data showed that AP amplitude was significantly decreased after capsaicin-induced intracellular calcium increase. In contrast, above treatments have no effects on depolarization or repolarization durations. 3. Spatiotemporal properties of action potential generated in primary sensory neuron can be modified under the state of inflammatory painPrevious investigation had shown that disequilibrium of calcium homeostasis caused plasticity of AP waveform. Then we asked the question whether AP waveform changed under pathological situations? The potential functional significance of AP plasticity was valuable to be uncovered. Then we used inflammatory pain model to investigate the plasticity of AP waveform under pathological situations.1) Behavioral tests demonstrated that paw withdrawal thermal latency (PWTL) and paw withdrawal mechanical threshold (PWTL) significantly decreased after subcutaneously injection of CFA (100μl, post 1 d) or melittin (100μg/100μl, post 2 h). In vivo electrophysiological recording tests demonstrated that noxious thermal and mechanical (brush, pressure and noxious pinch) stimuli cause increasing spike firing rates in spinal cord dorsal horn WDR neurons after CFA or melittin peripheral administration. The results showed that peripheral administration of CFA or melittin caused inflammatory pain.2) Effects of CFA or melittin on the excitabilities of DRG neurons. By using DiI ( 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate17 mg/ml,10μl dissolved in 10% DMSO)s.c. injection, we identified two classes of DRG neurons ( phasic neuron and tonic neuron) innervating the glabrous skin of the hind paw of rat. The data showed that CFA and melittin treatment had different effects on the excitabilities of two classes of DRG neurons. Tonic neuron increased excitabilities through lowering rheobases of AP generation and increasing the number of spike firing, whereas the excitabilities of phasic neuron were not changed by inflammation treatment. The data indicated that noxious information could be transmitted by both nonrhythmic and rhythmic activities of neural coding under inflammatory pain.3) Effects of CFA or melittin on spatial and temporal parameters of action potentials in two classes of DRG neurons. In tonic neuron, electrical stimuli-evoked(intensity, 50-500 pA; duration, 3 ms; interval, 200 ms) AP amplitude and overshoot were significantly increased, depolarization duration and depolarization duration were shortened after inflammation treatment by CFA or melittin. In phasic neuron, the effects of CFA and melittin were different. After CFA treatment, AP amplitude and overshoot were significantly increased, depolarization duration and repolarization durationare shortened. After melittin treatment , AP amplitude and overshoot were significantly increased, repolarization duration, but not depolarization duration were shortened. Above data indicated that,except for the changing of neuronal excitabilities, DRG neurons changed AP waveforms under inflammatory pain. The variability of AP waveform might be another novel activity of neuronal plasticity.4) Potential roles of TTX on AP plasticity DRG neuron under inflammatory pain. In DRG neurons of naive rat,TTX (1μM)significantly lengthened depolarization duration and decreased amplitude of AP evoked by electrical stimuli-evoked(intensity, 50-500 pA; duration, 3 ms; interval, 200 ms). However, the effects of TTX on of AP waveform (depolarization duration and amplitude) were significantly decreased after inflammation treatment by CFA (100μl, post 1 d) or melittin (100μg/100μl, post 2 h). Above data indicated that TTX-R sodium current components contributing to AP amplitude and depolarization were increased after peripheral inflammation treatment.4. Up-regulation of TIX-R sodium channels is one of important underlying mechanisms of spatial plasticity in primary sensory neurons1) Immunohistochemistry data showed that NaV1.8-positive staining was mainly in medium- and small-sized DRG neurons, whereas NaV1.9-positive staining was only in small-sized DRG neurons in naive rat. After CFA (100μl, post 1 d) or melittin (100μg/100μl, post 2 h) s.c.injection, the mean numbers of NaV1.8-positive neurons increased 93% and 50%, respectively. The mean numbers of NaV1.9-positive neurons increased 74% and 47%, respectively.2) Western blot data showed that both NaV1.8 and NaV1.9 proteins expressed in low levels in DRG naive rat. After CFA or melittin treatment, the relative intensities of NaV1.8 protein increased with 84.25% and 55.06%, respectively. NaV1.9 protein increased with 51.97% and 37.64%, respectively.3) RT-PCR data showed that both NaV1.8 and NaV1.9 gene expressed in low levels in DRG naive rat. After CFA or melittin treatment, the relative intensities of NaV1.8 gene increased with 116.02% and 82.01%, respectively. NaV1.9 gene increased with 87.86% and 43.36%, respectively. 4) Patch clamp data showed that after CFA or melittin treatment, the densitity of NaV1.8 current was significantly increased with 55.12% and 64.48%, respectively. The densitity of NaV1.9 current was also significantly increased with 87.08% and 70.30%, respectively.Above data indicated that up-regulation of NaV1.8 and NaV1.9 in gene and protein levels and facilitation of channel function were mainly responsible for the spatial plasticity of AP in DRG neuron.5. Pathological significance of spatiotemporal plasticity of primary sensory neuronal action potentials1) Immunohistochemistry and western blotdata showed that three days after twice daily i.t. administration (45μg) of fluorescence (Carboxyfluorescein,FAM)-labled antisense oligodeoxynucleotide (AS OND) to NaV1.8 and NaV1.9 could significantly decreased NaV1.8 or NaV1.9 up-regulation induced by CFA (100μl, post 1 d) or melittin (100μg/100μl, post 2 h) in both the number of positive neurons and protein levels. These data indicated that AS OND could significantly inhibited the NaV1.8 and NaV1.9 up-regulation induced by inflammation.2) Behavioral tests showed three days after twice daily i.t. administration (45μg) of AS OND to NaV1.8 and NaV1.9 had different effects on inflammatory pain induced by CFA (100μl, post 1 d) or melittin (100μg/100μl, post 2 h). Comparison with mismatch (MM) treatment, i.t. administration of NaV1.8 AS significantly inhibited CFA, but not melittin, -induced heat and mechanical hypersensitivity. Conversely , NaV1.9 AS treatment significantly inhibited heat hypersensitivity induced by melittin,but not by CFA. Above results indicated that NaV1.8 mainly contributed to heat and mechanical hypersensitivity induced by CFA-induced chronic inflammatory pain, whereas NaV1.9 mainly contributed to heat hypersensitivity induced by melittin-induced acute inflammatory pain.3) Patch clamp data showed that three days after twice daily i.t. administration (45μg) of AS OND to NaV1.8 and NaV1.9 had different effects on AP waveform generated in inflammatory pain induced by CFA (100μl, post 1 d) or melittin (100μg/100μl, post 2 h). NaV1.8 AS, but not NaV1.9 AS significantly inhibited AP amplitude and lengthened depolarization duration after CFA treatment. Conversely, NaV1.9 AS, but not NaV1.8 AS significantly inhibited AP amplitude and shortened depolarization duration after melittin treatment.Above data indicated that plasticity of AP waveform generated in DRG neurons had different molecular basis and was close relevant to the different pathological states of inflammatory pain. During the process of CFA-induced chronic inflammatory pain, NaV1.8 contributed to pain hypersensitivity through changing neuronal AP waveform. During the process of melittin-induced acute inflammatory pain, NaV1.9 contributed to pain hypersensitivity through changing neuronal AP spatial plasticity.Conclusions1. Under certain condition (such as, spontaneous discharge or constant stimulation), action potential (AP) waveform is"all or none". However, AP spatial parameter (amplitude) and temporal parameters (depolarization duration and repolarization duration) of DRG neuron are not identical given different stimulus intensities, demonstrating action potentials generated in primary sensory neuron perform spatiotemporal plasticity.2. Disequilibrium of calcium homeostasis around DRG neuron decreases AP amplitude and lengthens duration in some degree, demonstrating that calcium homeostasis is required for the maintainance of"all or none"property in primary sensory neuron.3. Under inflammatory pain, spatiotemporal plasticity of AP appears in two classes of DRG neurons. Tonic neuron increases excitabilities through lowering rheobases of AP generation and increasing the number of spike firing and contributes to the transmission of noxious information. However, phasic neuron showing no changes in excitabilities may transmit noxious information through APs with increased amplitude and shortened duration.4. Up-regulation of NaV1.8 and NaV1.9 in both gene and protein levels and facilitation of channel function are mainly responsible for the spatiotemporal plasticity of AP generated in DRG neuron.5. Spatial plasticity of AP generated in DRG neurons has different molecular basis and is closely relevant to the different pathological states of inflammatory pain. During the process of CFA-induced chronic inflammatory pain, NaV1.8 contributes to heat and mechanical hypersensitivity through changing neuronal AP spatial parameter. During the process of melittin-induced acute inflammatory pain, NaV1.9 contributes to heat hypersensitivity through changing neuronal AP AP spatial parameter.